| Trypsin Inhibitor (HWTX-Ⅺ) is peptide newly discovered from the spider Ornithortonus huwena (Wang). It consists of 55 amino acid residues, of which six cysteines have formed three pairs of disulfide bonds, relative molecular mass 6166.2Da. It belongs to BPTI/Kunitz-type serine protease inhibitor family, which is a stronger inhibitor for typsion, the inhibitory activity is more stronger than BPTI. Preliminary pharmacological experiments confirm that HWTX-Ⅺwould have a good treatment on acute animal pancreatitis models, indicating that HWTX-Ⅺcould be used to develop a new theropeutics for the diseases associated with trypsin.The expression of the recombinant HWTX-XI (rHWTX-Ⅺ) in prokaryotic expression system and Saccharomyces cerevisiae expression system had been successfully achieved in our laboratory, but these expression systems were limited to laboratory-scale. Pichia pastoris expression system, as a eukaryotic expression system, developed rapidly in recent years, can produce proteins with post translation modification and processing. Furthermore, this expression system can produce secreted proteins that could be purified easily, and would be very suitable for industrial-scale expansion. The HWTX-Ⅺgene was cloned by PCR by using the primers designed based on HWTX-ⅪcDNA gene, and then it was linked into the plasmid pPIC9K and transformed into Top1 OF’ bacteria. The recombinant plasmid pPIC9K-HWTX-Ⅺwas successfully constructed, as confirmed by the colony PCR method and sequencing Linearized pPIC9K-HWTX-Ⅺby Bg1Ⅱwas transformed into the host strain of yeast GS115. The multiple copy transformants GS115-pPIC9K-HWTX-Ⅺwould be obtained through G418 gradient screening of all the positive clones, which were identified as His+Muts phenotypes. The expression of the transformants GS115-pPIC9K-HWTX-Ⅺwas induced with 0.5% methanol concentration, and then expression supernatant was analyzed by Tricine-SDS-PAGE to confirm the target protein with molecular mass about 6.7KD. RHWTX-Ⅺwas purified by ion exchange chromatography and RP-HPLC. The purity of rHWTX-Ⅺwas more than 98% as revealed by analytical RP-HPLC. Its molecular weight was 6704 as determined mass spectrometry, which showed that it was the right expression product. We compared the inhibitory activity against trypsin of rHWTX-Ⅺwith the natural one through spectrophotometry method, showing that the Ki of rHWTX-Ⅺand the natural one were in the same order of magnitude. This means that HWTX-Ⅺwas correctly expressed, processed and secreted in Pichia pastor is.In this study, the optimization of the flask fermentation conditions of rHWTX-Ⅺexpression in the Pichia pastoris was also performed. We designed the orthogonal test of three factors inducing temperature, methanol concentration and the initial pH of medium at four different levels. Resultly, all the conditions above had a significant effect on the expression of rHWTX-Ⅺand the expression level was 26.5mg/ml under the optimized conditions (28℃, methanol 2%, pH4.0). In addition, we carried out a small-scale test in the fermentation tank on the basis of the shake-flask cultivation, attempting to explore the optimal conditions for fermentation from the fermentation medium and fermentation conditions to improve the expression level of rHWTX-Ⅺ.In conclusion, using recombinant Pichia pastoris can significantly increase the expression level of rHWTX-Ⅺ. This finding has laid the foundation on the research and development of acute pancreatitis drugs based on the HWTX-Ⅺas a precursor molecule. |
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